title: Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects.
abstract: Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using unlabeled AA and several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d(2)-AA and 10, 10, 13,13-d(4)-AA. The primary KIE for steady-state cyclooxygenase catalysis, (D)k(cat), ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9-2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes slow formation of a pentadienyl AA radical and rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of lipoxygenase indicates very different mechanism of hydrogen transfer.
authors.affiliation: Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.forename: Jian-Ming
authors.initials: JM
authors.lastname: Lü
authors.forename: Wilfred A
authors.initials: WA
authors.lastname: van der Donk
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kulmacz
authors.forename: Ah-lim
authors.initials: AL
authors.lastname: Tsai
mesh_terms: D016718:Arachidonic Acid / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D005609:Free Radicals; C408694:tyrosine radical; D016718:Arachidonic Acid; D014443:Tyrosine; D003903:Deuterium; D011451:Prostaglandin-Endoperoxide Synthases; D010100:Oxygen